Objective To investigate changes of serum levels of interleukin(IL)-4,-6,-8 and tumor necrosis factor-?(TNF-?),and probe their clinical significance in children with Henoch-Schonlein purpura(HSP)/ HenochSchonlein purpura nephritis(HSPN).Methods Serum levels of IL-4,-6,-8 and TNF-? of 45 children with HSP/HSPN and 43 healthy children were examined by enzyme linked immunosorbent assay(ELISA),changes and correlation between the aforementioned cytokines in children with HSP /HSPN were analyzed.Results 1.Serum levels of IL-4,-6,-8 and TNF-? of children with HSP were higher than those of control group(P0.05);The serum level of TNF-? was positively correlated to the serum levels of IL-6,-8 of patients with HSP(r=0.670 P

Objective To establish a molecular inversion probe ( MIP) method for detection of single base drug-resistance mutation in Hepatitis B virus ( HBV) gene.Methods The HBV wild type and YVDD mutant strain were isolated by Daping Hospital of the Third Military Medical University.The MIP was designed and applied to detect the HBV drug-resistance YVDD mutation in one case of wild type and one case of YVDD mutant HBV strain isolated previously.The results of MIP method were compared with that of sequencing to evaluate the detection accuracy.Results Thermal cycling single-base extension and connection reaction performed by Taq DNA Ligase and Ampligase DNA Ligase could ensure the specificity of the detection.The optimum probe concentration of MIP was 1 nmol/L.Through detection of the target gene with different DNA concentrations , the detection sensitivity of MIP was determined as 1 nmol/L.The results of MIP were consistent with that of sequencing method in detection of the clinical samples.Conclusion MIP is successfully used to detect single-base drug-resistance mutation in HBV gene.

Monitoring engraftment of donor cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is supposed to be important for the early diagnosis of graft failure or relapse of malignancy. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeats (STR) as markers are attractive because they are sensitive and can be performed rapidly. The intent of this study was to test a novel approach for assessment of donor engraftment using denaturing high-performance liquid chromatography (DHPLC) combined with STR-PCR. The feasibility of this assay and the accuracy of semi-quantitative results were tested in-vitro by using serial DNA mixtures from unrelated individuals. The results showed that dilution experiments of the mock chimerism sample revealed a clear correlation between the percentage of donor or recipient DNA and the proportion of allele peak areas, with the limit of detection for a minor DNA percentage being 5%. Discrimination between donor and recipient was possible in all patients analyzed (n = 51) except for 5 patients whose pre-transplant samples were not available and identical twins in one case. STR results were the same as values obtained by capillary electrophoresis combined with fluorescence labeling multiply PCR. Results were also compared with data obtained with FISH analysis in a subgroup of patients receiving grafts from sex-mismatched donors or with PCR-detectable disease-specific gene products analysis. The results of the microsatellite analysis correlated well with the corresponding clinical findings. Full donor chimerism (FDC) were detected in all patients; decreasing values of donor chimerism were detected concomitantly with the appearance of relapse of disease in 3 patients. Samples from eight patients receiving HLA mismatched-haploidentical transplants from related donors together with cord blood transplants from unrelated donors were analyzed by this method. The results showed all 8 patients achieved FDC derived from related donors. It is concluded that this novel approach allows a rapid, sensitive, economical, auto-mated and non-isotopic STR-PCR testing, thus provides a reliable alternative for assessment of the status of engraftment after allo-HSCT.
Adolescent ; Adult ; Child ; Child, Preschool ; Chromatography, High Pressure Liquid ; methods ; Female ; Graft Survival ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Leukemia ; genetics ; surgery ; Male ; Microsatellite Repeats ; genetics ; Middle Aged ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Transplantation, Homologous

To study feasibility of HLA-DRB1 matching by using denatured high performance liquid chromatography (DHPLC), 20 pairs of DNA samples from donors and recipients of hematopoietic cell transplantation (HCT) for DRB1 matching and 2 pairs of samples from donors and recipient of HCT for DRB1 mismatching were studied by DHPLC and PCR-SSP. After being amplified and annealed slowly to produce heteroduplex, PCR products for exon 2 of DRB1 were detected by DHPLC to find matched or mismatched peaks in chromatogram. The results showed that DHPLC and PCR-SSP were consistant with matched or mismatched HLA-DRB1 typing. The results of DHPLC and PCR-SSP for matching were compared by using kappa test (kappa = 0.776, P = 0.00), which suggested DHPLC for HLA-DRB1 matching was in agreement with PCR-SSP. In conclusion, DHPLC for HLA-DRB1 matching is economic and convenient, moreover, will not be affected by unknown genes in HLA-DRB1 locus.
Base Sequence ; Blood Donors ; Chromatography, High Pressure Liquid ; methods ; Feasibility Studies ; HLA-DR Antigens ; genetics ; immunology ; HLA-DRB1 Chains ; Hematopoietic Stem Cell Transplantation ; Histocompatibility Testing ; methods ; Humans ; Molecular Sequence Data ; Polymorphism, Genetic ; genetics

Objective To observe the therapeutic efficacy of hyperbaric oxygen (HBO) for focal cerebral infraction influenced by the treatment time after permanent middle cerebral artery occlusion (MCAO) in rabbits. Methods Seventy-five male rabbits were randomly divided into simple MCAO group (n=25), MCAO+HBO group (100% O2, 250 kPa, 1 h/d, from 1 d after MCAO, n=25) and MCAO+DHBO group (100% O2, 250 kPa, 1 h/d, from 7 d after MCAO, n=25). Behaviors and volumes of infarction were observed, and microdialysis was applied to monitor the concentrations of glucose, lactate, pymvate and glutamate around the infarct foci at 1, 3, 10 and 30 d after permanent MCAO. Results Behaviors'score was lower in MCAO+HBO group than the others (P<0.05). The infarct volume from day 3 to day 30 was significantly smaller in MCAO+HBO group than in the other 2 groups (P<0.05). The lactate and pyruvate ratio was increased after MCAO in three groups, but they were lower in the MCAO+HBO group than in the others at day 1 and day 3 (P<0.05). The glutamate concentration was increased after MCAO, peaked at 3 d, but at day 1 and day 3 the glutamate concentration was lower in the MCAO+HBO group (P<0.05). Conclusions HBO treatment could protect the brain from infarction through improving the energy metabolism and decreasing the excitatory amino acids disorders around the infarct foci after MCAO in rabbits. In order to improve the therapeutic efficacy of HBO, it should be performed as possible.

Objective To evaluate the safety and efficacy of endovascular stent placement in the management of ruptured vertebral artery dissecting ancurysms. Methods A retrospective analysis was conducted in 9 patients with ruptured vertebral artery dissecting aneurysms treated in our department between July, 2004 and January, 2007 by means of endovascular stenting. Of these cases, 3 were treated with exclusive stent placement and 6 with stent-assisted coiling. Results Stent deployment and coil embolization were carried out successfully in all the cases. The vertebral arteries with stent placement remained patent after the surgery, and stent thrombosis or aneurysmal rupture occurred in none of the cases. During the follow-up for 4 to 39 months, no clinical evidence of rebleeding was found in these patients. Follow-up angiography was performed in 6 of the patients 3-18 months after the surgery, and two patients treated with stent-assisted coiling showed aneurysm recurrence or enlargement and underwent secondary internal trapping or proximal occlusion; two patients receiving endovascular stenting showed complete embolization of the anenrysm, and one patient showed partial embolization. Conclusion Endovascular stent placement is safe and effective for management of raptured vertebral artery dissecting aneurysms and allows the involved artery to remain patent after the surgery. Close follow-up is necessitated in these patients, and the long-term effect of this treatment remains to be further observed.

OBJECTIVETo investigate the effects of ammonium perchlorate (AP) on thyroid functions and mRNA expression levels of thyroglobulin (Tg) and thyroperoxidase (TPO) genes of rats.

METHODSThirty SD male rats were randomly divided into six groups: control group, iodine-deficient group, low dose AP group (130 mg/kg), moderate dose AP group (260 mg/kg), high dose AP group (520 mg/kg) and high iodine-combined group. After the rats were exposed orally for 90 days, serum free-thyroxine (FT(4)), free-triiodothyronine (FT(3)) and thyroid stimulating hormone (TSH) were measured using radioimmunoassays. mRNA expression levels of thyroglobulin (Tg) and thyroperoxidase (TPO) genes were detected by real-time quantitative PCR.

RESULTSSerum FT(4) levels in moderate dose AP group and high dose AP group were [(9.540 ± 1.327) fmol/ml] and [(6.509 ± 1.949) fmol/ml] respectively, which were significantly lower than that [(13.505 ± 1.276) fmol /ml] in control group (P < 0.05 or P < 0.01). Serum TSH level in high dose AP group was [(1.227 ± 0.295) mIU/L], which was significantly higher than that [(0.545 ± 0.282) mIU/L] in control group (P < 0.05). The mRNA expression levels of thyroglobulin (Tg) gene in all groups exposed to AP were significantly lower than that in control group (P < 0.01). The mRNA expression level of thyroperoxidase (TPO) gene in high dose AP group was significantly higher than that in control group (P < 0.05).

CONCLUSIONAP can reduce the serum FT(3) and FT(4) levels of rats, increase the serum TSH level of rats and decrease obviously the mRNA expression levels of Tg and TPO genes. In addition, high iodine can reduce the toxic effects of AP on thyroid gland of rats to some extent.

Animals ; Iodide Peroxidase ; genetics ; metabolism ; Iodine ; administration & dosage ; Male ; Perchlorates ; toxicity ; Quaternary Ammonium Compounds ; toxicity ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Thyroglobulin ; genetics ; metabolism ; Thyroid Gland ; drug effects ; metabolism ; Thyrotropin ; blood ; Thyroxine ; blood ; Triiodothyronine ; blood

OBJECTIVETo explore the prophylaxis effect of pretreatment of allograft with methoxypolyethylene glycol-succinimidyl-propionic acid ester (mPEG-SPA) and anti-OX40L monoclonal antibody (McAb) on acute graft-versus-host disease (aGVHD) after allogeneic bone marrow transplantation (allo-BMT) in mice.

METHODSResponder splenocytes from C57BL/6 donor mice (H-2(b)) were co-cultured with stimulator splenocytes from BALB/c recipient mice (H-2(d)) for 7 days in the presence or absence of anti-OX40L McAb followed by mPEG-SPA chemical modification. Donor bone marrow cells plus the mixed culture of T-cells were then transplanted into lethally irradiated BALB/c mice. The BALB/c recipient mice were divided into four groups: group A (allo-BMT control group), group B(mPEG-SPA modification group), group C (anti-OX40L McAb pretreated group) and group D (mPEG-SPA and anti-OX40L McAb dual-treated group). Survival time and survival rate of the recipients were observed after allo-BMT. GVHD was assessed by clinical signs and histological changes of skin, liver and small intestines. Enzyme-linked immunosorbent assay (ELISA) was used to detect cytokines (IL-4, IL-10 and INF-gamma) production. Flow cytometry (FCM) analysis was used to detect allogeneic chimerism.

RESULTS(1) The mice in group A developed typical clinical signs of aGVHD and all mice died within 17 days after BMT with an average survival time (AST) of (12.1 +/- 5.5) days. The signs of aGVHD were less evident in mice of groups B, C and D, and their AST (36.2 +/- 24.9, 32.0 +/- 24.8 and 44.3 +/- 23.2 days, respectively) were all longer than that in group A (P < 0.05). AST of group D being the longest (P < 0.05). The survival rates at day 60 post-BMT in groups B, C and D were 50%, 41.7% and 66.7%, respectively. (2) Serum IFN-gamma level was increased after BMT in group A, and peaked in day 10 to day 15 post-BMT, while the level was decreased in groups B, C and D, reached the nadir on the day 10 post-BMT, with the lowest in group D (P < 0.01). After BMT, IL-4 and IL-10 levels were slightly decreased in group A, their levels were elevated in groups B and C (P < 0.05) and even more significantly increased in group D (P < 0.01). IL-4 and IL-10 levels peaked between day 10 and 15 post-BMT. (3) The average proportion of H-2(b) positive cells in recipient mice was 95% - 100% on day 60 post-BMT, with complete donor-type implantation.

CONCLUSIONCombination of mPEG-SPA and anti-OX40L McAb can block T-cell activated antigens and co-stimulatory pathway, regulate the T cells differentiation and induce the immune shift of Th0 cells toward Th2 cells. The immune tolerance induced by this method can significantly relieve aGVHD after allo-BMT.

Animals ; Bone Marrow Transplantation ; Graft vs Host Disease ; prevention & control ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Transplantation, Homologous

OBJECTIVEThe aim of the study is to explore the effect of NF-kappa B signal pathway in neonatal sepsis so as to provide the experimental base for corresponding clinical treatment of the sepsis, in which NF-kappa B is taken as the target.

METHODSThe sepsis model was established in newborn rats by giving Staphylococcus aureus subcutaneously: (1) The electrophoretic mobility shift assay (EMSA) was used to observe the activity of NF-kappa B in the lungs and the livers in newborn rats with Staphylococcus aureus sepsis. (2) Immunohistochemical method was used to observe the activity of NF-kappa B P56 in newborn rats with Staphylococcus aureus sepsis. (3) The anti-oxidant pyrrolidine dithiocarbamate (PDTC) was used to observe its effect on NF-kappa B activities of liver and lungs and on the activity of splenic NF-kappa B P56 in newborn rats with Staphylococcus aureus sepsis.

RESULTSIn newborn rats with Staphylococcus aureus sepsis, the NF-kappa B activity in lungs was enhanced at the 1st hour and reached to the peak level at the 3rd hour; then, it was weakened gradually and at the 24th hour faded away. The activity of the liver NF-kappa B was also activated and peaked at the 4th hour; then, it was gradually weakened and at the 24th hour faded away. The positive expression of splenic NF-kappa B P56 began to be intensified at the 1st hour (12.0 +/- 3.7), peaked at the 3rd hour (51.4 +/- 5.9) and showed insignificant differences at the 24th hour (3.4 +/- 1.4) as compared with the sepsis group. PDTC had an inhibitive effect on the activities of liver NF-kappa B and lung NF-kappa B and on the positive expression of splenic NF-kappa B P56 used in the dosage of 50-200 mg/kg. The larger the dosage was used, the more intensified inhibitive effect could be obtained. In the dosage of 200 mg/kg, the inhibitive effect was the most intensified.

CONCLUSIONS(1) In newborn rats with Staphylococcus aureus sepsis, the NF-kappa B of lungs, liver and spleen were activated, and all indicate a peak. (2) The anti-oxidant PDTC can inhibit NF-kappa B activity in a dose-effect fashion in newborn rats with Staphylococcus aureus sepsis.

Animals ; Animals, Newborn ; Antioxidants ; therapeutic use ; Dose-Response Relationship, Drug ; Electrophoretic Mobility Shift Assay ; Liver ; drug effects ; metabolism ; Lung ; drug effects ; metabolism ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Pyrrolidines ; therapeutic use ; Rats ; Rats, Wistar ; Sepsis ; metabolism ; Staphylococcus aureus ; pathogenicity ; Thiocarbamates ; therapeutic use